Screening And Analysis Of Proteins Which Interact With CDF1Protein In Rice

A new rice flowering-time regulated gene CDF1(Constitutive Delayed Flowering1) was cloned by screening and identifying T-DNA insert mutant materials. CDF1encoded an E3ubiquitin ligase containing C3HC4RING-finger domain, cdfl mutant exhibited a phenotype of late flowering-time both under short-day and long-day condition. This study focus on the molecular regulatory networks of rice heading-time in which CDF1gene involved. We used the yeast two-hybrid and microarray technology to identify the interacting proteins of CDF1protein and the genes regulated by CDF1gene. Meanwhile, to elucidate the function of these genes in rice flowering regulation network, we took advantage of domestic and foreign rice mutant resources. The main results of this study are as follows:1. The heading-time and field phenotype of cdfl material were observed, and the genotype of cdfl material was identified by genomic primers and T-DNA primer. The results indicated that the delayed phenotype co-segregated with the T-DNA homologous insertion, which consistent with the previous results.2. The ds RNAi vector of CDF1gene was constructed, and the positive transgenic plants were obtained by Agrobacterium-mediated transformation method.3. According to the results of gene microarray, we selected a total of74differentially expressed genes. Using RT-PCR and real-time PCR to detect the expression levels of these genes in cdfl mutant and wild-type plant found that the expression levels of31genes were decreased,26genes were increased and9genes didn’t change obviously in cdfl mutant; besides, there were8genes haven’t detected any results. These data indicated that the majority of testing results were consistent with the gene microarray results, and many flowering-regulatory genes were regulated by CDF1gene.4.22pGBKT7vectors that fused different CDF1truncated proteins were constructed. The autoactivation of these truncated proteins were tested by yeast transformation experiments, which confirmed that the181-215amino acid region of CDF1protein have transcriptional activity. 5. Total RNA of penultimate leaf were respectively isolated from the cultivar Zhonghuall at10:00P.M. and9:30A.M. which before its flowering transition period (35th day after sowing). The two Y2H cDNA library of rice penultimate leaf was generated by recombination-mediated SMART technology in yeast strain Y187and AH109, respectively. The titers, transformation efficiency and recombinant rate of the two cDNA library were suitable the experimental requirements.6. We screened the cDNA library for three times, meanwhile the correctness of the screening results were verified by using yeast co-transformation technology. We obtained21putative interacting proteins with CDF1(99-619) by screening10:00P.M. cDNA library; while obtained22and21interacting proteins with CDFl(288-667) by screening10:00P.M. and9:30A.M. cDNA library, respectively. A total of54different interacting-proteins were obtained by screening cDNA library in three repeats.7. The interaction relationship between CDF1protein and other proteins were explored by using yeast co-transformation experiment, the results indicated that CDF1protein interact with OsFMl protein and itself.8. Employing TAIL-PCR and APCR method to isolate the flanking sequences of22mutants, I obtained17flanking sequences, among them,15insert sites were correct. However, the results of co-segregation analysis showed that the mutant phenotype didn’t co-segregated with its genotype, so these insertion sites were not the cause for the mutant phenotype in each lines.